Glutamate is a signaling molecule that plays an important role in the normal physiological function of gastrointestinal tract including histamine-induced acid secretion in stomach, and contractility of the stomach and intestine. As an intracellular messenger, glutamate is involved in glucose-induced insulin exocytosis in pancreatic (-cells and glucagon exocytosis in pancreatic beta cells. The functional glutamaterigic systems have been characterized in digestive organs including stomach, intestine, and pancreas. Glutamate uptake into the secretory granules by vesicular glutamate transporter is a rate-limiting step for glutamate release. Our laboratory has recently cloned and functionally characterized a neuronal vesicular glutamate transporter (VGLUT2) that is expressed in pancreatic alpha and beta cells. The long-term goal of our laboratory is to study the regulation of vesicular glutamate transporter in digestive system. The primary purpose of this proposal is to determine the mechanisms of glucose-induced regulation of vesicular glutamate transporter gene expression in pancreas. The hypothesis to be tested in this proposal is that chronic regulation of VGLUT2 in beta and alpha cells, by high and low concentrations of glucose, respectively, is via transcriptional mechanisms. This hypothesis is supported by strong preliminary data including (i) high glucose concentration (12.8 mM) increases vesicular glutamate transport in ( cells and low glucose concentration (2.8 mM) increases vesicular glutamate transport in beta cells, (ii) VGLUT2 mRNA expression is increased by high glucose concentration in beta cells and by low glucose concentration in alpha cells, and the changes of mRNA expression can be blocked by actinomycin D, (iii) VGLUT2 mRNA is increased in genetic mouse model of non-insulin-dependent diabetes. We propose to study the regulation of VGLUT2 by three specific aims. First, characterize the transcriptional mechanism of VGLUT2 in response to changes of extracellular glucose concentration. Second, characterize VGLUT2 promoter and identify glucose-response cis-acting regulatory elements. Third, identify trans-acting protein factors involved in glucose-induced regulation of VGLUT2 and determine their functional role in the regulation of VGLUT2. The results of this study should provide fundamental information on the regulation of VGLUT2 by glucose, and on its functional roles in glucose--induced insulin and glucagon exocytosis in the pancreas.